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The peripheral T-cell lymphomas (PTCLs) are uniquely sensitive to epigenetic modifiers. Based on the synergism between histone deacetylase inhibitors and hypomethylating agents that we established in preclinical PTCL models, we conducted a phase 1 study of oral 5-azacytidine (AZA) and romidepsin (ROMI) in patients with advanced lymphoid malignancies, with emphasis on PTCL. According to a 3 + 3 design, patients were assigned to 1 of 7 cohorts with AZA doses ranging from 100 mg daily on days 1 to 14 to 300 mg daily on days 1 to 21, ROMI doses ranging from 10 mg/m2 on days 8 and 15 to 14 mg/m2 on days 8, 15, and 22, with cycles of 21 to 35 days. Coprimary end points included maximum tolerated dose (MTD) and dose-limiting toxicity (DLT). We treated a total of 31 patients. The MTD was AZA 300 mg on days 1 to 14 and ROMI 14 mg/m2 on days 8, 15, and 22 on a 35-day cycle. DLTs included grade 4 thrombocytopenia, prolonged grade 3 thrombocytopenia, grade 4 neutropenia, and pleural effusion. There were no treatment-related deaths. The combination was substantially more active in patients with PTCL than in those with non-T-cell lymphoma. The overall response rate in all, non-T-cell, and T-cell lymphoma patients was 32%, 10%, and 73%, respectively, and the complete response rates were 23%, 5%, and 55%, respectively. We did not find an association between response and level of demethylation or tumor mutational profile. This study establishes that combined epigenetic modifiers are potently active in PTCL patients. This trial was registered at www.clinicaltrials.gov as NCT01998035.

Peripheral T-cell lymphomas (PTCL) are a group of rare malignancies characterized by chemotherapy resistance and poor prognosis. Romidepsin and pralatrexate were approved by the US Food and Drug Administration for patients with relapsed/refractory PTCL, exhibiting response rates of 25% and 29% respectively. Based on synergy in preclinical models of PTCL, we initiated a phase 1 study of pralatrexate plus romidepsin in patients with relapsed/refractory lymphoma. This was a single institution dose-escalation study of pralatrexate plus romidepsin designed to determine the dose-limiting toxicities (DLTs), maximum tolerated dose, pharmacokinetic profile, and response rates. Patients were treated with pralatrexate (10 to 25 mg/m2) and romidepsin (12 to 14 mg/m2) on 1 of 3 schedules: every week × 3 every 28 days, every week × 2 every 21 days, and every other week every 28 days. Treatment continued until progression, withdrawal of consent, or medical necessity. Twenty-nine patients were enrolled and evaluable for toxicity. Coadministration of pralatrexate and romidepsin was safe, well tolerated, with 3 DLTs across all schedules (grade 3 oral mucositis × 2; grade 4 sepsis × 1). The recommended phase 2 dose was defined as pralatrexate 25 mg/m2 and romidepsin 12 mg/m2 every other week. Twenty-three patients were evaluable for response. The overall response rate was 57% (13/23) across all patients and 71% (10/14) in PTCL. The phase 1 study of pralatrexate plus romidepsin resulted in a high response rate in patients with previously treated PTCL. A phase 2 study in PTCL will determine the efficacy of the combination. This trial was registered at www.clinicaltrials.gov as #NCT01947140.

Options for patients with relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL) after brentuximab vedotin (Bv) and autologous stem cell transplantation (ASCT) are limited. Immune checkpoint inhibitors (ICI) are active in this population but rarely induce complete response (CR). Ten patients with R/R cHL after ASCT and Bv received pembrolizumab (n = 8) or nivolumab (n = 2). Five had been previously exposed to 5-azacitidine on a phase 1 study. Among nine evaluable patients, seven (78%) achieved CR, one partial response, and one reduction of tumor burden. All five patients who had received 5-azacitidine prior to ICI achieved CR, while only two of four who did not receive prior 5-azacitidine achieved CR. At a median follow-up of 9.9 months [0.5–14.3], eight patients are alive and five are still receiving treatment. We documented an unprecedented CR rate after ICI in patients with R/R cHL. We hypothesize that hypomethylating agents might have an immune priming effect and enhance the efficacy of ICI. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0363-1) contains supplementary material, which is available to authorized users.

Our group has demonstrated that combinations of epigenetic modifiers produce potent synergy in pre-clinical models of PTCL, and has confirmed this data in early phase clinical studies. We showed that the combination of romidepsin and hypomethylating agents, decitabine or azacytidine, uniquely induces the expression of genes, including cancer testis antigens, gamma interferon and endogenous retrovirus. We are conducting a phase 1 study of pembrolizumab combined with pralatrexate alone (Arm A), with pralatrexate + decitabine (Arm B), or decitabine alone (Arm C) in patients with peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma. In addition, we are conducting a Phase 1/2A study of durvalumab combined with oral 5-azacitidine + romidepsin (Arm A), pralatrexate + romidepsin (Arm B), romidepsin (Arm C), or oral 5-azacitidine (Arm D) in patients with PTCL. Patient characteristics are listed in Table 1. We have treated a total of 12 patients across the 2 trials and 4 different Arms including pralatrexate plus pembrolizumab (3 patients), pralatrexate, decitabine and pembrolizumab (3 patients), decitabine and pembrolizumab (3 patients) and azacytidine, romidepsin and durvalumab (3 patients). Twelve patients out of 12 received at least one dose of drug and were evaluable for toxicity. There was a dose limiting toxicity (DLT) for each arm including prolonged grade 3 thrombocytopenia, febrile neutropenia, grade 3 hyponatremia and rash, and cytokine release syndrome, respectively. There were no treatment-related deaths. Eight patients out of 12 are evaluable for response at the time of this analysis. Three out of 8 patients achieved a complete remission, 3 out of 8 patients had a partial remission, and 2 out of 8 patients experienced progression of disease. Interestingly, all of the responses were seen in the triple combination of pralatrexate, decitabine, pembrolizumab and azacytidine, romidepsin and durvalumab. These preliminary clinical data suggest that the addition of immune-checkpoint inhibitors on an epigenetic backbone is safe and demonstrates encouraging responses in patient with PTCL and CTCL. These trials are registered at www.clinicaltrials.gov (NCT03240211, NCT03161223). Patient characteristics (n=12)Median age (range)68 (26-77)Sex, n (%) Male Female6 (50) 6 (50)Race, n (%)White/Non-Hispanic White/Hispanic Black Asian Unknown5 (42) 1 (8) 3 (25) 1 (8) 2 (17)Histology, n (%) PTCL, NOS AITL Mycosis Fungoides PTCL, NOS TFH ATLL Sezary Syndrome ALCL, ALK+2 (17) 2 (17) 2 (17) 1 (8) 1 (8) 1 (8) 1 (8)Stage at diagnosis IB II III IV Unknown1 (8) 2 (17) 3 (25) 4 (33) 2 (17)Median number of prior therapies (range) **2 unknown3 (0-5) Citation Format: Enrica Marchi, Helen Ma, Francesca Montanari, Ahmed Sawas, Jennifer K. Lue, Changchun Deng, Kareema T. Whitfield, Sandra Klein, Matthew V. Matthew, Freedy J. Loffredo, Luigi Scotto, Hyejung Lee, Hye A. Kim, Alice T. Jacobs, Aishling M. Rada, Karen A. Khan, Salvia Salvia, John Lister, Nora N. Benanni, Mark A. Francescone, Pier Luigi Zinzani, Wonseog Kim, Owen A. O'Connor. The integration of PD1 blockade with epigenetic therapy is highly active and safe in heavily treated patients with T-cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT160.

[This corrects the article DOI: 10.1155/2018/8573105.].

Bing–Neel syndrome is a rare manifestation of Waldenström macroglobulinemia characterized by lymphoplasmacytic cells’ infiltration into the central nervous system. We present a case of a 74-year-old patient with a known diagnosis of Waldenström macroglobulinemia and newly depressed consciousness. Flow cytology of his cerebral spinal fluid demonstrated a lambda light chain-restricted population of B-cells consistent with a CD5+ CD10+ B-cell lymphoma. Magnetic resonance imaging suggested involvement of the left optic nerve sheath and the bilateral orbital and parietal parenchyma and leptomeninges. He was diagnosed with Bing–Neel syndrome and treated with intrathecal liposomal cytarabine, intravenous high-dose methotrexate, and rituximab without improvement. Subsequently, he started treatment with ibrutinib 560 mg daily and concurrent rituximab. Within three months, he showed clinical and radiologic improvement. The patient has continued on ibrutinib and has now been stable for over 36 months. This represents the longest reported period of successful treatment of Bing–Neel syndrome with ibrutinib.

Introduction: Natural Killer cells (NK cells) play an important role in tumor immune-surveillance. NK-mediated cell killing can occur through distinct mechanisms: (1) by activating the receptor NKG2D and natural cytotoxicity receptors (NCR), and (2) through the potent activating receptor CD16 (FcγRIII) which mediates antibody-dependent cell-mediated cytotoxicity (ADCC). Therapeutic strategies to harness the ability of NK cells to induce an ADCC response are emerging. AFM13 is a CD30/CD16A targeting high affinity bispecific tetravalent antibody that engages and activates NK cells. This study evaluates the ability of AFM13 to engage innate immunity through specific activation and recruitment of NK cells to tumors expressing CD30 and the impact of these effects on clinical outcome. In addition, it examines the immunologic changes in the tumor and peripheral blood as a function of the dose and method of administration of AFM13 over time. Methods: Subjects with relapsed or refractory CD30 expressing lymphoma with cutaneous involvement were recruited into 3 treatment cohorts: (1) 1.5 mg/kg IV weekly, (2) 7 mg/kg IV weekly and (3) 7 mg/kg continuous intravenous infusion (CIVI) over 5 days weekly. Each cohort consisted of 3 patients. Subjects received 8 weeks of therapy each cycle. Response assessment was performed on week 11 of each cycle by mSWAT, photography, PET imaging and peripheral blood flowcytometry. A second cycle was administered if there was no progression of disease. Subjects underwent mandatory tumor biopsies and peripheral blood immunologic studies during the first cycle of therapy as follows: pretreatment, day 5 post first dose, week 4 and week 8 of therapy. An optional biopsy was also collected at end of study. Tumor biopsies were analyzed and evaluated by a pathologist and IHC image analyzer to characterize immune cell subpopulations. Peripheral blood samples were analyzed by flowcytometry. Results: Nine subjects were accrued. Their age ranged from 37-79 years, 3 of 9 where white and 6 of 9 were men. The median number of prior therapies was 4 (1-11), 2 patients had progressed on Brentuximab vedotin, and 5 patients received total skin electron beam radiotherapy. The disease etiologies were as follows: 4 patients with transformed mycosis fungoides, 2 patients with systemic anaplastic large cell lymphoma-ALK negative, 2 patients with non- transformed mycosis fungoides and 1 patient with cutaneous anaplastic large cell lymphoma. Among the 8 subjects who were evaluable for response 4 of 8 (50%) achieved an objective response rate (ORR). In cohort 1 we observed 1 complete response (CR), 1 partial response (PR) and 1 stable disease (SD). In cohort 2, we observed 3 SD. In cohort 3, we observed 2 PRs and one subject awaiting disease assessment. Infusion related reaction (IRR) were observed in 4 of 9 subjects and managed conservatively. Cellulitis was noted in 3 subjects of which 2 developed bacteremia requiring intravenous antibiotics, and was not considered related to AFM13 treatment. There were no other treatment-emergent adverse events. In the peripheral blood, we observed a decrease in circulating NK cells during therapy (Weeks 1- 8) with post therapy recovery (by week 11) by following cells that were CD56+ CD3- , CD56+ CD16+ and NKp46+ by Flow Cytometry. We observed an increase over time in the expression of the activation marker CD69 on NK cells from responders compared to non-responders. Similarly, tumor biopsies in responders showed increased infiltration of CD56+ NK cells as opposed to non-responders. Flow quantitation of circulating CD4+ CD25+ T cells (Tregs) shows a decrease in responders vs. non-responders. Conclusions: AFM13 demonstrated a high ORR of 50 % among a population of heavily pretreated patients with a CD30 positive lymphoproliferative T-cell malignancy. AFM 13 exhibited activity post Brentuximab vedotin failure. In addition, biological changes in NK cells may predict response. This data are the first to demonstrate the therapeutic advantages of this bispecific, and the first to correlate changes in NK cells as a function of dose and schedule. AFM13 is able to engage the innate immune system through a specific activation and recruitment of circulating NK cells to tumors expressing CD30. This immunological tumor response may correlate with clinical response. We have begun to expand the patient cohorts, and pursue additional comprehensive correlative studies in this population. Disclosures Rodig: KITE: Research Funding; Bristol Myers Squibb: Research Funding; Affimed: Research Funding; Merck: Research Funding. O'Connor:Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding; Celgene: Research Funding.

Introduction: PTCL may represent the prototypical epigenetic malignant disorder. First, recurring mutations in important epigenetic regulators, such as TET2, IDH2, and DNMT3, have been described across several PTCL subtypes, though especially in AITL. Second, HDACi have only been approved as single agents in patients with relapsed/refractory (R/R) PTCL, and exhibit T-cell lineage-specific activity across disease subtypes. Thirdly, preclinical evidence from our group suggests marked class synergism between HDACi and HMA. In addition, across a panel of diverse T-cell lymphoma lines, the combination of HDACi and HMA induced the expression of many cancer testis antigens and genes involved in the interferon pathway/viral antigen response. Based on this collective experience, a phase 1/2 trial of romidepsin (ROMI) and oral 5-azacitidine (AZA) was launched to determine if this activity is reproduced in patients with R/R PTCL. Methods: Patients with R/R lymphoma were eligible for the phase 1, whereas the phase 2 only enrolled patients with PTCL, and included both R/R and treatment-naïve individuals. The dose-escalation portion of the study followed a 3+3 design with progressively increasing dose intensity across 7 cohorts. The phase 1 primary objectives were determination of the maximum tolerated dose and dose-limiting toxicity. Phase 2 primary objectives were overall response rate (ORR, i.e., complete response [CR] + partial response [PR]), progression free survival, and duration of response (DOR). Results: 36 patients have been enrolled to date, 26 in phase 1 and 10 in phase 2. In the intention-to-treat population the median age was 56 years [23-83] with 59% being males. Twelve patients had Hodgkin lymphoma, 8 had a B-cell lymphoma, and 16 had a T-cell lymphoma (6 enrolled in the phase 1 and 10 in phase 2). The median number of prior therapies was 6 [1-15] for the phase 1 population and 1 [0-6] for the phase 2. After a median of 2 cycles [0-16], all phase 1 patients have discontinued therapy, whereas 6 out of 10 patients in phase 2 are still on treatment after a median of 2.5 cycles [1-14]. No patient discontinued therapy due to adverse events (AE). The most frequent hematologic G3-4 AE included neutropenia (39%), lymphopenia (39%), and thrombocytopenia (28%). The most frequent non-hematologic G3-4 AE included febrile neutropenia (8%), hyponatremia (5%), and lung infection (5%). Other common G1-2 toxicities included hyperglycemia, hypoalbuminemia, nausea/vomiting, and fatigue. The recommended phase 2 dose for the combination was declared AZA 300 mg days 1-14 and ROMI 14 mg/m2 days 8, 15, and 22 on a 35-day cycle. Thirty-two patients are evaluable for response. Of these, 27 had measurable disease. The ORR and CR in the overall population are 41% and 22%, respectively. However, while only 2 (11%) patients with non-T-cell lymphoma responded, of which one was a CR, 11 of 14 (79%) patients with T-cell lymphoma responded, including 6 (43%) who attained CR (see waterfall plot in figure). Notably, all 6 evaluable patients with AITL responded, and 3 achieved a CR. Two of these 3 achieved PR before reaching CR. Responses have been durable and the median DOR has not been reached [0.2-13.1+ months]. The AUC for ROMI at 10 mg/m2 (N = 15) and 14 mg/m2 (N = 20) were 2021.5 +/- 1461.3 h*ng/mL and 1565.7 +/- 5656.2 h*ng/mL, respectively, with a median half-life of 4.8 and 4.9 hours respectively, which is comparable to single-agent values. Conclusions: The combination of AZA and ROMI is well tolerated, with cytopenias being the most common G3-4 AE. The combination appears to exhibit marked T-cell lineage-specific activity. The 100% ORR in AITL patients is unprecedented and warrants detailed follow-up. Ongoing sequencing analysis will evaluate the impact of recurring mutations on the clinical activity of the combination. The study is actively accruing (NCT01998035). Figure Figure. Disclosures O'Connor: ADC Therapeutics: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding.

To learn more about the process of amyloid beta-protein (Abeta) deposition in the brain, human prefrontal cortices were fractionated by sucrose density gradient centrifugation, and the Abeta content in each fraction was quantified by a two-site enzyme-linked immunosorbent assay. The fractionation protocol revealed two pools of insoluble Abeta. One corresponded to a low-density membrane domain; the other was primarily composed of extracellular Abeta deposits in those cases in which Abeta accumulated to significant levels. Abeta42 levels in the low-density membrane domain were proportional to the extent of total Abeta42 accumulation, which is known to correlate well with overall amyloid burden. In PDAPP mice that form senile plaques and accumulate Abeta in a similar manner to aging humans, Abeta42 accumulation in the low-density membrane domain also increased as Abeta deposition progressed with aging. These observations indicate that the Abeta42 associated with low-density membrane domains is tightly coupled with the process of extracellular Abeta deposition.

The PDAPP transgenic mouse, which overexpresses human amyloid precursor protein (APP717V→F), has been shown to develop much of the pathology associated with Alzheimer disease. In this report, levels of APP and its amyloidogenic metabolites were measured in brain regions of transgenic mice between 4 and 18 months of age. While absolute levels of APP expression likely contribute to the rate of amyloid β-peptide (Aβ) deposition, regionally specific factors also seem important, as homozygotic mice express APP levels in pathologically unaffected regions in excess of that measured in certain amyloid plaque-prone regions of heterozygotic mice. Regional levels of APP and APP-β were nearly constant at all ages, while Aβ levels dramatically and predictably increased in brain regions undergoing histochemically confirmed amyloidosis, most notably in the cortex and hippocampus. In hippocampus, Aβ concentrations increase 17-fold between the ages of 4 and 8 months, and by 18 months of age are over 500-fold that at 4 months, reaching an average level in excess of 20 nmol of Aβ per g of tissue. Aβ 1–42 constitutes the vast majority of the depositing Aβ species. The similarities observed between the PDAPP mouse and human Alzheimer disease with regard to Aβ 42 deposition occurring in a temporally and regionally specific fashion further validate the use of the model in understanding processes related to the disease.

Background Clinical studies of β-amyloid (Aβ) immunotherapy in Alzheimer's disease (AD) patients have demonstrated reduction of central Aβ plaque by positron emission tomography (PET) imaging and the appearance of amyloid-related imaging abnormalities (ARIA). To better understand the relationship between ARIA and the pathophysiology of AD, we undertook a series of studies in PDAPP mice evaluating vascular alterations in the context of central Aβ pathology and after anti-Aβ immunotherapy. Methods We analyzed PDAPP mice treated with either 3 mg/kg/week of 3D6, the murine form of bapineuzumab, or isotype control antibodies for periods ranging from 1 to 36 weeks and evaluated the vascular alterations in the context of Aβ pathology and after anti-Aβ immunotherapy. The number of mice in each treatment group ranged from 26 to 39 and a total of 345 animals were analyzed. Results The central vasculature displayed morphological abnormalities associated with vascular Aβ deposits. Treatment with 3D6 antibody induced clearance of vascular Aβ that was spatially and temporally associated with a transient increase in microhemorrhage and in capillary Aβ deposition. Microhemorrhage resolved over a time period that was associated with a recovery of vascular morphology and a decrease in capillary Aβ accumulation. Conclusions These data suggest that vascular leakage events, such as microhemorrhage, may be related to the removal of vascular Aβ. With continued treatment, this initial susceptibility period is followed by restoration of vascular morphology and reduced vulnerability to further vascular leakage events. The data collectively suggested a vascular amyloid clearance model of ARIA, which accounts for the currently known risk factors for the incidence of ARIA in clinical studies.

Background: Patients with relapsed/refractory (R/R) classical Hodgkin lymphoma (cHL) after brentuximab vedotin (Bv) and autologous stem cell transplantation (ASCT) represent an unmet medical need. Allogeneic SCT can be curative, but is burdened with significant morbidity and mortality. Nivolumab and pembrolizumab are anti-programmed death (PD)-1 monoclonal antibodies and are referred to as immune checkpoint inhibitors (ICI). These agents have shown remarkable activity in patients with R/R cHL, but rarely induce complete response (CR). Studies on ovarian cancer cell lines have shown that exposure of tumor cells to the hypomethylating agent (HMA) 5-azacitidine, may induce transcription of latent endogenous retroviral genes, which then trigger a T-cell-mediated immune response. Therefore, HMA can act as "immunologic primers" and enhance the efficacy of ICI. We reviewed our experience with ICI in patients with R/R cHL, and compared responses between those who had been previously exposed to 5-azacitidine to those who had not. Methods: Twelve patients with R/R cHL received pembrolizumab at the dose of 2 mg/kg every 3 weeks (N = 9), or nivolumab at the dose of 3 mg/kg every 2 weeks (N = 3). Response was assessed by positron emission tomography/computerized tomography. For patients who were transplant candidate, ICI were used as a bridge to the procedure. Results: The patient median age was 35 years [range 25-79]. The median number of prior treatments was 11 [range 3-16] and 75% of patients had had ≥ 7 lines of therapy. All patients had received ASCT and Bv. Six had been exposed to 5-azacitidine for a median of 2.5 months [range 2-16] as part of a phase 1 study (NCT01998035). Four of them had received it immediately prior to ICI, the other two within 14 months of starting ICI. A total of 141 patient-courses were completed, with a median of 7 courses per patient [range 1-39]. There were 6 grade ≥ 3 adverse events (AE): infusion reaction (N = 1), thrombocytopenia (N = 1), grade-5 respiratory failure (N = 1), myelodysplastic syndrome, (N = 2, one of which pre-existent, both evolved into fatal acute myeloid leukemia (AML)), and acute kidney injury (N = 1, pre-existent). Grade 1-2 AE included infusion reaction (5), elevated thyroid stimulating hormone levels (3), diarrhea (2), fatigue (1). Ten patients are evaluable for response: 7 (70%) achieved CR, one partial response, one a reduction of tumor burden and one stable disease. Five of the 6 patients who received 5-azacitidine prior to ICI achieved CR, while only 2 of 4 who did not receive prior 5-azacitidine achieved CR. One patient's evaluation is pending at this time. At a median follow-up of 14.2 months [range 0.5-17.7], 9 patients are alive and 7 are still receiving treatment. Two patients with radiographic progression continue to be treated given sustained clinical benefit. One patient transitioned to allogeneic SCT while in complete remission and one discontinued due to symptomatic progression. The median progression-free survival has not been reached as of this writing (7.9+ months). Conclusions: We report the largest cohort of patients treated with ICI after being exposed to HMA. In these patients with very heavily pretreated cHL we observed a higher-than-expected CRR. Most serious AE were not related to ICI therapy. The finding that most CR clustered in patients previously exposed to 5-azacitidine may reflect synergism between HMA and ICI and support the notion of HMA-mediated "immunologic priming". This hypothesis is being tested in prospective clinical trials at our institution. Disclosures Sawas: Gilead Sciences: Speakers Bureau; Seattle Genetics: Honoraria. Amengual:Acetylon Pharmaceuticals: Research Funding; Bristol-Myers Squibb: Research Funding. O'Connor:Spectrum: Research Funding; Spectrum: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; TG Therapeutics: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Celgene: Research Funding.

2552Background: Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of lymphomas in which only 20-25% of patients experience long-term survival with CHOP chemotherapy. Recently several drugs...

Background: Cerebral amyloid angiopathy is a common neuropathological feature of Alzheimer’s disease and is characterized by vascular deposition of fibrillar amyloid -protein (VA ). Vascular structural changes are associated with VA deposits, including localized loss of smooth muscle cells (SMC) and changes in extracellular matrix (ECM). Passive immunization with antibodies to the A N-terminus (3D6, aa 1-5) has been shown to effectively reduce amyloid deposition in the vasculature of PDAPP transgenic mice. In the present study we characterized structural changes induced by amyloid on SMC and ECM of PDAPP mouse vessels and assessed the effects of passive immunotherapy. Methods: Mice were immunized weekly for either 3 or 9 months with 1 or 3 mg/Kg of 3D6 antibody. High-resolution, quantitative IHC analyses of vascular components ( -actin for SMC and collagen-IV for ECM) were performed on meningeal vessels from the sagittal sinus, where VA deposition is prominent ( 70% of vessels affected). Microhemorrhage events were monitored by hemosiderin detection or ferritin immunohistochemistry. Results: We found that changes in the vascular wall are invariably associated with VA , and they included both degeneration (decreased thickness) and hyperplasia/hypertrophy (increased thickness) of SMC and ECM. These two contrasting findings were often observed in the same vessel and were not present in wild type animals or PDAPP vessels lacking amyloid. The extreme degrees of thickening and thinning of the SM resulted in a widely variable vascular phenotype in untreated PDAPP mice. Passive immunotherapy restored the pattern of vascular SMC and ECM thicknesses and reduced the phenotypic variability in a doseand time-dependent manner, with the high dose of 3D6 reaching control levels (wild type) at 9 months. Although the incidence of microhemorrhage increased in the 3-month group, it reduced to control levels after 9 months of treatment. Conclusions: Our results suggest that passive immunotherapy allows the recovery of meningeal vessels from amyloid-induced structural changes. Furthermore, the treatment-related increase in microhemorrhage appears to be a transient event that resolves during VA clearance. Mechanisms of repair may be triggered by VA removal, which ultimately lead to recovery from vascular dysfunction. Experiments to assess this possibility are in progress.

Background: In vivo administration of antibodies against the amyloid-β (Aβ) peptide has been shown to reduce and reverse the progressive amyloidosis that develops in a variety of mouse models of Alzheimer’s disease (AD). This work has been extended to clinical trials where subsequent autopsy cases of AD subjects immunized against Aβ showed similar reductions in parenchymal amyloid plaques, suggesting this approach to reduce neuropathology in man is feasible. Objective: Multiple hypotheses have been advanced to explain how anti-Aβ antibodies may lower amyloid burden. In this report, we compare approaches utilizing either plaque-binding or peptide-capturing anti-Aβ antibodies for effectiveness in reducing amyloidosis in a mouse model of AD. Methods: A plaque-binding monoclonal antibody (3D6) and an Aβ peptide-capturing monoclonal antibody (266) were compared in chronic treatment and prevention paradigms using a transgenic mouse model of AD. The effects of antibody therapy on plaque burden and plasma clearance of Aβ were investigated by quantitative imaging and clearance studies of intravenously injected 125I-Aβ. Results: The plaque-binding antibody 3D6 was highly effective in either treatment or prevention of amyloidosis. In these studies, the peptide-capture antibody 266 showed no reduction in amyloidosis in either paradigm and showed trends towards increasing amyloidosis. Antibody 266 was also found to greatly prolong (>180-fold) the normally rapid peripheral clearance of Aβ, in contrast to that found with 3D6 (>24-fold). Conclusion: Reversing and preventing Alzheimer’s type amyloidosis is most effectively accomplished with anti-amyloid antibodies that avidly bind plaque.

Passive immunotherapy with amyloid(A ) antibodies reduces a number of AD pathologies including parenchymal A plaques, neuritic dystrophy, synaptic loss, and memory function in mouse models, and many of these effects are beginning to be seen in AD patients. In addition to parenchymal deposition of amyloid, AD is also characterized by amyloid fibrils in the cerebral vasculature in the majority of patients, a condition called cerebral amyloid angiopathy (CAA). Previous studies have suggested that immunotherapy does not clear and may increase CAA and the incidence of microhemorrhage. In the present study we demonstrate that certain immunotherapies are surprisingly highly efficacious in removing CAA and that the incidence of microhemorrhage can be modulated by dosage. We examined the effects of passive immunization on CAA in 2 PDAPP mouse studies: one a comparison of antibodies to different A epitopes (3D6, A 1-5; 12A11 and 10D5, A 3-7; 266 A 16-23) at one dose, and a 3D6 dose-response study at 3 doses. CAA clearance and microhemorrhage incidence were assessed after 6 months using A IHC and hemosiderin detection.

Transgenic mice mimicking certain features of Alzheimer's disease (AD)-pathology, namely amyloid plaques and neurofibrillary tangles, have been developed in an effort to better understand the mechanism leading to the formation of these characteristic cerebral lesions. More recently, these animal models have been widely used to investigate emergent therapies aimed at the reduction of the cerebral amyloid load. Several studies have shown that immunotherapy targeting the amyloid peptide (Abeta) is efficacious at clearing the amyloid plaques or preventing their formation, and at reducing the memory/behavior impairment observed in these animals. In AD, different types of plaques likely have different pathogenic significance, and further characterization of plaque pathology in the PDAPP transgenic mice would enhance the evaluation of potential therapeutics. In the present study, a morphological classification of amyloid plaques present in the brains of PDAPP mice was established by using Thioflavin-S staining. Neuritic dystrophy associated with amyloid plaques was also investigated. Finally, the efficacy of passive immunization with anti-Abeta antibodies on the clearance of Thio-S positive amyloid plaques was studied. Our results show that distinct morphological types of plaques are differentially cleared depending upon the isotype of the antibody.

In AD certain brain structures contain a pathological density of A beta protein deposited into plaques. The effect of genetic mutations found in early onset AD patients was an overproduction of A beta 42, strongly suggesting that overproduction of A beta 42 is associated with AD. We hypothesized that an immunological response to A beta 42 might alter its turnover and metabolism. Young PDAPP transgenic mice were immunized with A beta 1-42, which essentially prevented amyloid deposition; astrocytosis was dramatically reduced and there was reduction in A beta-induced inflammatory response as well. A beta 1-42 immunization also appeared to arrest the progression of amyloidosis in older PDAPP mice. A beta immunization appears to increase clearance of amyloid plaques, and may therefore be a novel and effective approach for the treatment of AD.

One hallmark of Alzheimer disease is the accumulation of amyloid beta-peptide in the brain and its deposition as plaques. Mice transgenic for an amyloid beta precursor protein (APP) mini-gene driven by a platelet-derived (PD) growth factor promoter (PDAPP mice), which overexpress one of the disease-linked mutant forms of the human amyloid precursor protein, show many of the pathological features of Alzheimer disease, including extensive deposition of extracellular amyloid plaques, astrocytosis and neuritic dystrophy. Active immunization of PDAPP mice with human amyloid beta-peptide reduces plaque burden and its associated pathologies. Several hypotheses have been proposed regarding the mechanism of this response. Here we report that peripheral administration of antibodies against amyloid beta-peptide, was sufficient to reduce amyloid burden. Despite their relatively modest serum levels, the passively administered antibodies were able to enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid. When examined in an ex vivo assay with sections of PDAPP or Alzheimer disease brain tissue, antibodies against amyloid beta-peptide triggered microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation. These results indicate that antibodies can cross the blood-brain barrier to act directly in the central nervous system and should be considered as a therapeutic approach for the treatment of Alzheimer disease and other neurological disorders.

Publisher Summary This chapter shows that PDAPP mice over expressing a mutation associated with some cases of familial early onset AD express several of the major pathological hallmarks associated with AD. Amyloid plaques in PDAPP mice appear quite similiar to A/3 deposits in AD as shown by a variety of different antibodies and stains, and are of both the diffuse and compacted varieties. Additionally, a subset of these amyloid plaques appears to be neuritic plaques. Neurodegenerative changes, including the loss of synaptic and dendritic proteins, abnormal phosphorylation of cytoskeletal elements, subcellular degenerative changes, and the deposition of lysosoma1 and acute phase proteins has also been seen in PDAPP mouse brains. Reactive astrocytosis and microgliosis have also been observed in association with the amyloid plaques in the PDAPP mice. No neurofibrillary tangles or paired helical filaments have been found in the mice to date. It remains unknown whether mice are capable of generating these in a manner comparable to AD in less than two years. Extensive behavioral analyses are currently being performed in these mice, and preliminary results indicate that the PDAPP mice are significantly impaired on a variety of different learning and memory tests. In conclusion, the PDAPP mouse model doesn't display all the pathological hallmarks of AD, but it does display most of them in a robust manner that increases with age and gene dosage. Therefore, this transgenic model provides evidence that alterations in APP processing and AB production can result in AD-like neuropathology, can contribute to a mechanistic understanding of AD, as well as providing a useful animal model for the testing of various therapeutic interventions directed toward specific aspects of the neurodegenerative process.